Pneumocystis is a major opportunistic pathogen of immunocompromised patients. Because Pneumocystis cannot be reliably cultured, molecular approaches have been utilized to identify and characterize antigens of this organism. Recombinant antigens can then be used to examine host immune responses to Pneumocystis infections. We have an ongoing project to characterize the antigens of mouse, rat, and human Pneumocystis. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. It is necessary to use Pneumocystis from both sources because the organisms are different species. Subsequently, we identified a number of clones from a cDNA library of rat Pneumocystis that contain genes encoding for the MSG. These clones are clearly related but not identical, demonstrating that multiple genes encode the MSG. We have continued studies to characterize potential antigens of Pneumocystis. We have cloned a number of human Pneumocystis MSG genes and have expressed a full-length MSG in two fragments. We developed an ELISA to examine antibody responses to these antigens, and have utilized it to examine sera from patients with or without HIV infection, and with or without a history of PCP, as well as sera from a variety of healthy conrols. In about 15% of healthy patients followed serially we have been able to document changes in antibody titers, suggesting that these individuals have developed reinfection or reactivation of Pneumocystis infection. We will continue these studies to better understand the epidemiology of Pneumocystis infection in humans. We have also identified the unique expression site of MSG in human Pneumocystis, and can now identify the MSG variants that are expressed in a patient with PCP. Within this expression site we have identified a region of tandem repeats that varies among different Pneumocystis isolates, and thus provides a new method for typing human Pneumocystis. We are also studying a protein related to kexin, a protease of yeast, that appears to be an antigen of rodent pneumocystis. We have cloned and expressed recombinant kexin from human pneumocystis and are currently examining immune responses to the recombinant protein. The goal of this study is to better understand the pathogenesis of Pneumocystis pneumonia with the hope that we can use this information to control or prevent this disease.